CD97 was discovered as a result of "reverse genetics" and therefore, relatively little is known about the mechanisms involved in CD97 function. The primary structure suggests a G-protein coupled receptor (GPCR), which utilizes an adhesion class of ligand. We have determined that integrin alpha 5, beta 1 is one binding protein for CD97, and conversely CD97 can act as a ligand to stimulate endothelial cell activation via integrin receptor binding. We recently have discovered that ectopic CD97 leads to specific heterotrimeric G protein activation. Having identified a signaling pathway activated by CD97, it has been possible to assay CD97-binding proteins for signal transducing functions. We have demonstrated that intercellular integrin alpha 5 beta 1 presentation to CD97-expressing cells activates a specific class of heterotrimeric G protein. However, presentation of CD55, another CD97-binding protein, is neither sufficient nor required for such activation. This is the first data that directly shows a heterotrimeric G protein mechanism of signaling for an adhesion class GPCR. Because CD97 is a GPCR, it is a good candidate as a target for small molecule inhibitors capable of "locking" CD97 in an inactive state. We are developing high throughput assays to identify small molecules capable of initiating or blocking activation of CD97 using a signal transduction-dependent read-out. We anticipate that the identification of such small molecules may be of use in future drug development for the prevention or treatment of metastasis. In order to address the function of CD97 relative to cancer biology, we have used several approaches that assess invasiveness and tumorigenicity. CD97 is expressed in thyroid, colon, and prostate cancers but not in the normal epithelial counterparts. Reducing CD97 expression in prostate cancer cell lines inhibits their migration and invasion in vitro. Using an anaplastic thyroid cancer cell line that expresses an intermediate level of CD97, we have determined that increasing CD97 expression leads to enhanced motility and invasiveness in vitro. Reducing the level of CD97 greatly diminishes the tumorigenic potential in xenograft assays. Thus, CD97 may be a factor that contributes to the grade of thyroid carcinoma. We are further testing the effect of CD97 expression on the progression of follicular thyroid carcinomas in vivo using transgenic mouse models. We are combing thyrocyte-specific CD97 expression with tumor-promoting genetic lesions to investigate effects upon thyroid cancer development and progression. Different lines of RET/PTC1 transgenic mice develop follicular thyroid hyperplasia or noninvasive papillary thyroid carcinomas. Similarly, mice carrying homozygous mutation in the thyroid hormone receptor develop follicular thyroid carcinomas with a weakly penetrant invasive phenotype. We have developed transgenic strains of mice that overexpress CD97 in thyrocytes. By crossing appropriate transgenic strains, we will evaluate an effect of CD97 overexpression in thyrocytes predisposed to develop thyroid cancer. Cohorts of mice are being analyzed for the level of invasive thyroid cancer, comparing CD97-expressing to non-expressing animals at 6, 12, and 15 months of age.